The detection of biological and pharmaceutical substances is a crucial aspect of research and clinical medicine. At present, each substance requires a separate assay, and these separate assays vary in their specificity and sensitivity. For some substances there is no currently available appropriate assay. Immunologic means are used to detect substances by those present assays which are among the most sensitive and specific. This technology is dependent upon the molecular interaction between an antibody, which is a protein that will specifically recognize and bind to a biological or pharmaceutical substance, which is known as the antigen or hapten. An antigen is a substance which is capable of producing an antibody. A hapten is a substance which alone is not capable of producing an antibody but when bound to a carrier which is an antigen is capable of producing an antibody directed against itself. Antibodies have particular regions or molecular domains, called binding domains, that are active in recognizing the antigen or hapten. It is not necessary to have the entire antibody molecule present, but only one of these binding domains in order to form a complex.
The degree of specificity in the recognition and binding of an antibody and an antigen or hapten is dependent upon the extent of uniqueness of the antigen's or hapten's 3-dimensional molecular structure and chemical properties and the extent of the correlation between those properties and the antibody's structure and properties. Two different antigens or haptens may have some aspect of their structure in common, and this can lead to the phenomenon of cross reactivity where the same antibody will recognize both antigens or haptens. To minimize this problem, it is important that the antibodies be as specific as possible. The recent development of hybridoma technology allows the selection for antibodies of high specificity. These antibodies have been employed in clinical assays for a few select antigens, with each test being for only one antigen. A particular research or clinical problem may indicate a need for testing for several antigens, thus requiring several separate assays.
The principle of such immunological assays is to detect the presence of a complex formed by the interaction of an antigen and an antibody. Current technology uses one of several methods to detect these complexes. One method employs a second antibody which is labeled either radioactively or enzymatically and which is directed against the same antigen. This will interact with the complex by binding to the antigen, sandwiching it between the two antibodies. These complexes are separated from unbound antigen or antibody by centrifugation if the assay is done in a liquid phase or serial washings if the assay is done on a solid phase. The presence of label is interpreted as a positive result and can be quantitated.
Another method uses highly purified antigen that is labeled and which competes with the test antigen for binding to the antibody in forming a complex. These complexes are separated from unbound antigen or antibody as described above. In this method the absence of label is interpreted as a positive result and can be quantitated. This method relies upon a constant supply of highly purified labeled antigen, as well as the highly specific antibody required by the first method.
A third approach, crossed immunoelectrophoresis (CIE), involves the formation of antigen-antibody complexes in a three dimensional matrix, with the visualization of these complexes using stains. This method requires substantially more antibody and antigen than the other methods and the utilization of special high voltage electrical equipment. It is therefore more expensive and cumbersome than other methods, but is able to resolve complex mixtures of antigens.